The measurement of the refractive index or RI of a fluid such as a gas or liquid has widespread applications across many industries. RI is a property of a fluid which characterizes its response to an externally applied electromagnetic radiation field. Different substances respond to the same field to an extent dependent upon the specific material and it is this varying response which forms the basis for both quantifying a given material and distinguishing it from another. Generally though, the qualitative aspects of an RI measurement are less sought after than its appeal as a quantitative tool since many substances, such as sugars, are less amenable to other forms of analysis such as UV absorbance detection (i.e., they lack of strong UV chromophore) or fluorescence. RI is sometimes referred to as a universal detector since so many substances will exhibit an RI response. In particular, an RI detector preceded by a separation means, such as a liquid chromatograph, will yield responses for virtually all substances. In this measurement mode, a sample containing one or more analytes is injected onto a chromatographic column. Subsequent continuous flow of a clean mobile phase through the column leads to a separation in time of the individual analytes. These analytes elute or exit from the column as individual peaks having a characteristic volume and retention time reflective of the analyte and column packing material. The peak is transported from the column to the RI detector, which produces a response proportional to analyte concentration. Since each peak contains the original quantity of analyte dissolved within the mobile phase, the more compact or narrow the volume of the peak, the larger the RI response will be for the same injected mass. Compact peaks are characteristic of minimal dispersion during transit of the analyte peak from the column to the detector. The process whereby the peak exiting the column is broadened during transport to a downstream detector is generally referred to as post-column dispersion.
Proper management of post-column dispersion can allow the volumetric scale of the separation to be decreased, which can yield meaningful gains in signal enhancement for many detection methods, including concentration sensitive analyzers such as differential RI detectors. Peak volumes decrease in proportion to the cross-sectional area of the column. Thus, for two columns whose diameters differ by a factor of two, the peak volume for the smaller ID column is expected to be four times smaller and therefore for the same mass injected, the concentration should be four times larger. There are other important advantages in going to smaller scale separations. For example, reduced solvent consumption is an advantage for applications that employ expensive mobile phases, which are common in RI detection.
Large scale chromatographic systems can be categorized as those employing separation columns with internal diameters (IDs) greater than about 4 mm, small scale columns with IDs in the range of about 1-4 mm, and capillary scale systems with IDs less than about 1 mm. Chromatographic theory can predict the peak volume of a retained analyte and it is this volume which serves as a guide in judging the effects of post-column dispersion. For the preceding range of columns, packed with conventional particles, typical peak volumes for early eluting analytes (k′=2) are shown in Table 1.
TABLE 1Time, min,ColumnParticlePeakOptimumfor 1ColumnLength,Size,VolumeFlow Rate,ColumnID, mmmmmicrons(4.4%), μLml/minVolume4.61503.51800.562.943.01001.7440.490.950.51001.71.20.0140.95
In practice, system parameters such as flow rate, operating pressure, etc. will be affected by the column choice. Relative to a large scale separation, the same application can be carried out with a small scale system in a manner that yields benefits both in time and reduced solvent consumption. As peak volumes for small scale systems are smaller, tighter constraints are placed on controlling sources of post-column dispersion. Accordingly, there is a need for low dispersion differential refractometers intended for separations conducted on small scale systems.
A broad range of RI detectors coupled to a separation system have been described in the art. For example U.S. Pat. No. 3,674,373 describes a heat exchanger for a differential refractometer. As is well-known, the temperature coefficient of the refractive index of most fluids is such that poor thermal control can lead to unwanted detector responses which are many times larger than the signal of interest. The '373 patent discloses tubing with inner diameters in the range of 0.02″ to 0.04″ with lengths of up to 12″. These tubing dimensions correspond to post-column volumes from 60 μL, to 160 μL, which are unsuitable for small scale separations. U.S. Pat. No. 3,999,856 describes a diffractometric refractometer which measures a phase shift between a probe beam which has passed through a reference and sample flow cell chamber. Flow cell volumes as small as 2 μL, are discussed, but such small cells generally have short mechanical pathlengths, which can lead to limitations when attempting to measure both very small and large refractive index differences. The '856 patent does not disclose detector volumes between the column and flow cell or thermal management of the sample or reference streams.
Many techniques have been described in the art for measuring refractive index difference based upon a phase shift of light which has traveled through reference and sample fluid cells and which is then recombined in a plane distant from the cell. These techniques, broadly classified as interferometric methods, can be carried out with low volume flow cells but still require low pre-cell fluidic volumes and good thermal management to enable accurate RI differences.
U.S. Pat. No. 4,952,055 describes a beam displacement technique carried out in a capillary-based flow cell. While low volume cells are feasible, a setup method is described that requires alignment of the probe beam to the flow cell at an angle based upon the refractive index of the cell material (glass) and the sample fluid. Thus, measuring RI differences over a large range of absolute RI (e.g., from 1.30 to 1.60 RI units) as would be necessary in a general purpose RI detector, would necessitate optical realignments which could negatively impact instrument performance. Other techniques, such as those employing evanescent sensing (e.g., as disclosed in U.S. Pat. No. 5,311,274) may also be realized in low volume configurations but have limited range due to the dependence upon the refractive index of the light-carrying material.
U.S. Pat. No. 5,606,412 and U.S. Pat. No. 5,900,152 describe apparatus for modifying flow profiles within a non-circular flow cell by generally directing this flow towards the interior side surfaces of the cells. The apparatus of these patents refer to flow cells having volumes in the range of about 7 to 50 μL, which are more suitable for large scale chromatography.
Accordingly, there remains a need for robust, wide-ranging, and sensitive differential RI detectors exhibiting low dispersion.